Immunogens comprising the non-lytic membrane spanning domain of bacteriophages MS2 or PhiX174

ABSTRACT

The invention concerns a carrier-bound recombinant protein obtainable by expression of a fusion protein gene in gram-negative bacteria which codes for at least one hydrophobic non-lytically active protein domain capable of membrane integration as well as the recombinant protein and of a gene which codes for a lytically active membrane protein from bacteriophages or a lytically active toxin release gene or lytically active partial sequences thereof and isolation of the carrier-bound recombinant protein from the culture broth. The recombinant protein is thereby firmly incorporated into the cell wall complex of gram-negative bacteria via a target sequence. Furthermore the invention concerns a recombinant DNA for the production of the protein, the production process as well as the use of carrier-bound recombinant proteins according to the present invention for immunization and as vaccines.

The invention concerns carrier-bound recombinant proteins, a process for their production and their use, in particular as immunogens and vaccines.

The main purpose of the immunological system in humans and animals is to resist and avoid pathological damages which arise as a result of degenerate cells of infectious viruses, bacteria, fungi or protozoa. A special characteristic of the immunological system is that an increasingly stronger resistance occurs after repeated infections with pathogens. The aim of immunization is to build up the power of the immunological system against particular pathogens without causing corresponding diseases.

Antibodies and cellular T and B lymphocytes are responsible for the specific resistance to pathogens. An essential prerequisite for this is the recognition of foreign structures such as e.g. those which occur on a bacterial cell. Depending on the stimulation of the immunological system a temporary or a lifelong immunity to pathogens can be built up in this process after immunization.

It is important for the quality of monoclonal and polyclonal antibodies as well as for the effectiveness of vaccines that the immunological response to the antigen occurs to a sufficient extent. However, viral antigens or recombinant human proteins often show a poor immunological response or none at all if they are used without further modification. For this reason these antigens are often linked to carriers (preferably to proteins) in order to amplify the immunological response. However, the antigens can be changed at or near the antigenic determinants by the binding of the antigens to the carrier. As a result the immunological response can be substantially weakened.

In order to improve the immunological response it is advantageous to incorporate such antigens into the outer membrane of bacteria and to use these complexes as immunogens (J. Immunol. 139 (1987) 1658-1664, Bacterial Vaccines and Local Immunity--Ann. Sclavor 1986, n. 1-2, pp. 19-22, Proceedings of Sclavo International Conference, Siena, Italy, 17-19 November 1986). Attenuated or dead pathogens (bacteria or viruses), treated partial components of pathogens (membrane proteins of bacteria, structural proteins of viruses) or recombinant live vaccines (viruses or bacteria) are also used.

A disadvantage of using live bacteria or viruses as immunogens for the immunization is that an undesired pathogenic spread of the germs cannot be excluded.

However, the antigenic determinant can be altered by killing or fragmenting the bacteria and viruses before their use as an immunogen or vaccine which can substantially reduce the immunological response.

The object of the present invention is therefore to provide immunogens and vaccines which do not have these disadvantages.

This object is achieved via a carrier bound, recombinant protein. This carrier bound, recombinant protein is obtained by expressing a first gene coding for a fusion protein and a second gene, hereinafter referred to as "lysis gene", which codes for one of: (a) a lytically-active, bacteriophage membrane protein, (b) a lytically-active, toxin release gene, or (c) a lytically active, partial sequence of one of these. The fusion protein comprises at least one hydrophobic, non-lytically active protein domain capable of membrane integration and a recombinant protein carrier bound, recombinant protein is then isolated from the culture broth.

The expression of the fusion protein gene and the lysis gene is preferably controlled by two different promoters (FIG. 1). The expression of the lysis gene is preferably delayed with respect to the expression of the fusion protein.

With this type of expression of fusion protein gene and lysis gene one obtains at first the integration of a multitude of fusion proteins into the membrane of the gram-negative bacteria used as the host organism and subsequently lysis of these bacteria takes place. The usually impermeable cell wall complex of the bacteria is made so permeable by this that the cytoplasmic components of the bacteria are released (Eur. J. Biochem. 180 (1989), 393-398). The morphology of the cells, for example the rod-form of E. coli cells, is preserved. A tunnel structure is merely formed in a localized area of the membrane. The tunnel formation is accompanied by a fusion of the inner and outer membrane at the edge of the tunnel. The bacterial coats formed in this way represent the carriers for the recombinant protein and are hereinafter denoted bacterial ghosts (FIG. 2 ).

The bacterial ghosts consist of a cytoplasmic (inner) membrane, periplasmic space and outer membrane whereby the integrity of the cell wall complex is preserved to a large extent. In the case of bacterial strains which have an additional S-layer coat (paracrystalline protein layer outside the outer membrane) this protein layer is also a component of the bacterial ghosts (Ann. Rev. Microbiol. 37 (1983), 311-339). The bacterial ghosts are therefore carriers of the recombinant proteins (immunogens) and, as a result of their composition (peptidoglycan, lipopolysaccharide), they at the same time constitute the adjuvant for amplifying the immunological response.

All gram-negative bacteria, preferably gram-negative pathogens such as e.g. Escherichia coli, Bordetella pertussis, Campylobacter jejuni, Corynebacterium diphteriae, Legionella pneumophilia, Listeria monocytogenes, Pseudomonas aeruginosa, Shigella dysenteriae, Vibrio cholerae, Yersinia enterolitica are suitable as host organisms (Schaechter, M., H. Medoff, D. Schlesinger, Mechanisms of Microbial Disease. Williams and Wilkins, Baltimore (1989)).

The carrier-bound recombinant proteins according to the present invention are surprisingly well suited as immunogens which results in pronounced immunological responses and very high antibody titres.

A particular advantage results from the fact that the recombinant protein is integrated into the membrane of the bacteria directly after the expression and thus the carrier binding is formed. As a consequence it is unnecessary to isolate the recombinant protein as such before production of the immunogen. Moreover, since it is sufficient for the production of bacterial ghosts containing immunogens when from several hundred up to the maximum possible number (ca. 50000) of recombinant antigens are integrated into the membrane of the bacterial ghosts, an over-expression of the recombinant protein is not necessary.

A further advantage of the process according to the present invention is that very many antigenic epitopes are presented in the cell wall complex of the bacterial ghosts. It has turned out that the target sequences for the recombinant proteins prefer certain regions within the bacterial cell wall complex for integration. These regions mainly constitute adhesion sites of the inner and outer membrane and are associated with the cell division of the bacteria. As a result the recombinant protein is not distributed uniformly but rather islet-type accumulations occur within the cell wall complex (cf. FIG. 2d). The clustered arrangement of the recombinant proteins within a relatively small region (cluster) has the advantage that the proliferation of B cells carrying immunoglobulin is stimulated. On the other hand the lipopolysaccharide present in the bacterial ghosts acts as a mitogen and also triggers a signal for the cell division. As a result one achieves an effective stimulation of the B-cell specific production of immunoglobulins.

In addition it has also turned out that the carrier-bound recombinant proteins according to the present invention are integrated into the bacterial membrane in their natural protein structures and thus in an active form.

This is particularly surprising since recombinant proteins are usually obtained in an inactive form as inclusion bodies (cf. EP-A- 0219 874, WO 89/03711) after expression in prokaryotes and can only subsequently be converted into the active form by denaturation and renaturation.

All proteins familiar to one skilled in the art are suitable as recombinant proteins. Human proteins and antigens, in particular viral antigens, are particularly preferably used. Their size is not limited. The molecular weight of the antigens is, however, preferably 2000 to 200000 Daltons.

The recombinant antigen has particularly preferably antigenic structures of human viruses and retroviruses such as e.g. of HIV (human immunodeficiency virus), HBV (hepatitis B virus), and EBV (Epstein Barr Virus)

The hydrophobic non-lytically active protein domains capable of membrane integration are hereinafter denoted target sequences. Complete sequences or partial sequences of membrane proteins which can, however, also be modified by amino acid substitutions are preferred as target sequences. Such a substitution should not, however, alter the structure of the corresponding protein.

Target sequences which are preferably used are those which--in contrast to the signal sequences of other membrane proteins--are not cleaved by proteases which are present in the membrane (e.g. signal peptidase and proteases of the periplasmic space). Target sequences can for example be derived from naturally occurring sequences of the lysis gene of the PhiXl74 phage group (for N-terminal targeting) as well as from the naturally occurring sequences of the lysis gene of the MS2 phage group (for C-terminal targeting) by protein engineering.

A hydrophobic alpha-helical protein domain consisting of 14 to 20 amino acids, which can be flanked N- and C-terminally by 2 to 30 arbitrary amino acids each, is preferred as the target sequence. At least one further protein domain can preferably be bound to this protein domain. The binding preferably takes place via flexible linker sequences. Flexible linker sequences are understood as hydrophilic amino acid sequences with 2 to 100 amino acids, preferably with 2 to 30 amino acids and with a disordered secondary structure (turn and random coil sequences).

The additional protein domains which are coupled to the first protein domain can be structured in an analogous manner to the first protein domain. It is, however, preferable that at least one of the additional domains posesses a β-pleated sheet structure and is composed of 10 to 16 amino acids, preferably 11 to 13 amino acids. The construction and secondary structure of such β-pleated sheet structures is preferably similar to amphipathic protein sequences which occur in porins of the outer membranes. For a N-terminal targeting it is preferable to use those target sequences which contain the amino acids 1 to 54 of protein E from the phage PhiXl74 (hereinafter denoted E' sequence) and which do not act lytically. For a C-terminal targeting it is preferable to use target sequences which contain the amino acids 21 to 75 of protein L from the phage MS2 (hereinafter denoted L' sequence) and which do not act lytically (for sequences compare EP-A 0 291 021). Sequences which are derived from the above-mentioned sequences of the E and L target sequences by a homologous amino acid substitution which does not cause an alteration in the secondary structure of the protein are also suitable.

Membrane proteins of bacteriophages are preferably understood as membrane proteins from bacteriophages of the Microviridae class, preferably from icosahedral phages, lytic phages and phages containing ssDNA, which can infect Enterobacteriacae. Examples of these are the phages PhiXl74, S13, G4, G6, G14, PhiA, PhiB, PhiC, PhiR which can infect E. coli C strains. Alpha 3 which can infect E. coli C and E. coli B strains is also suitable. The phages K9, St-1, PhiK, PhiXtB and U3 which can infect E. coli K12 strains are also suitable (Sinsheimer R. L. (1968) in: Prog. Nucl. Acid Res. Mol. Biol. (Davidson J. N. & Cohn W. W., eds) Vol.8, Academic Press, New York & London, pp. 115-169; Tessman E. S. & Tessmann I. (1978) in: The single-stranded DNA Phages (Denhardt D. T., Dressler D. & Ray D. S., eds.) Cold Spring Harbor Press, Cold Spring Harbor, pp. 9-29; Hayashi M., Aoyama A., Richardson D. L. & Hayashi M. N. (1987) in: The Bacteriophages, pp. 1-71).

Lysis proteins from the mentioned bacteriophages as well as other toxin release genes such as the colicin lysis gene (Microbiol. Sciences 1 (1984) 168-175 and 203-205) are preferably suitable as lytically active membrane proteins.

In a further, preferred embodiment, a binding partner for the recombinant protein is bound to it. This binding partner binds non-covalently. Examples of recombinant protein/binding partner pairs include, e.g., biotin and (strept)avidin, hapten and antibody, antigen and antibody, concanavalin and antibody, sugar and lectin, hapten and binding protein (e.g., thyroxin binding globulin and thyroxin), and oligopeptide and antibody. Additional substances may, in turn, be bound to the binding partner, either covalently or non-covalently.

Streptavidin, or avidin, and biotin are preferably used as the binding pair. Streptavidin or avidin is especially preferably used as the immobilized recombinant protein and biotinylated antigen is bound to it.

Furthermore, it is preferred that a protein be used as the recombinant protein which recognizes a chemical ligand. Examples for this are β-galactosidase/p-aminophenyl-β-D-thiogalactoside (a structural analogue of lactose), Gene 29 (1984) 27-31. Such substituted products are bound to the bacterial ghosts by the recognition of the active centre of the β-galactosidase without cleavage of the substrate.

The invention also concerns a recombinant DNA which contains a first DNA sequence (DNA target sequence), which codes for at least one hydrophobic non-lytically active protein domain capable of membrane integration, a second DNA sequence (DNA protein sequence) which codes for a recombinant protein, as well as a DNA sequence (DNA lysis gene) which is under separate control from this which codes for a lytically active membrane protein from bacteriophages or a lytically active toxin release gene or for their lytically active parts.

DNA sequences are preferred as DNA target sequences which code for the L' protein or the E' protein. DNA sequences are also suitable which code for amino acid sequences which are derived from these proteins having the same secondary structure. These sequences are preferably connected by DNA sequences which code for hydrophilic protein domains having 2 to 30 amino acids and a disordered secondary structure.

In a preferred embodiment the DNA lysis sequence contains the DNA sequence of the E protein, the DNA sequence of the L protein or the DNA sequence of the EL hybrid protein (for sequences cf. EP-A 0 291 021). Partial sequences thereof which act lytically are also suitable.

The DNA protein sequence is preferably the DNA sequence of a viral antigen (e.g. HIV, HBV, EBV) or of a recombinant human protein.

The invention also concerns a process for the production of a carrier-bound, recombinant protein which is characterized in that a fusion protein which contains at least one hydrophobic non-lytically active protein domain capable of membrane integration as well as a recombinant protein, and a lytically active membrane protein from bacteriophages or a lytically active toxin release gene or lytically active partial sequences thereof are expressed in gram-negative bacteria and the carrier-bound, recombinant protein is isolated from the culture broth. The transformation and expression can be carried out according to processes familiar to one skilled in the art. The transformation is preferably carried out by electroporation or conjugation.

During the fermentation the activity of the lytic protein is preferably at first inhibited or the expression of the lysis gene is repressed and the inhibition or repression is only abolished at a desired time, preferably in the late logarithmic phase.

In a further preferred embodiment the carrier-bound recombinant protein obtained in this way is incubated with a binding partner for the protein which is derivatised, if desired, and the conjugate which is formed is isolated. The above-mentioned partners of the binding pairs are suitable as the binding partner.

In a further preferred embodiment the genes of at least two different recombinant proteins are expressed according to the present invention. In this way immunogens or vaccines can be obtained which have several antigenic structures. In this connection it is particularly preferred to use the antigenic determinants of different viruses or retroviruses (e.g. HIV1, HIV2, HBV and EBV) as the recombinant proteins. For the expression these genes can be arranged in an expression vector either as an open reading frame in the 3' direction after the gene for the target sequence or a special vector can be used for each of the recombinant proteins to be expressed. In this case it is, however, necessary that the vectors are each provided with separate origins of replication and separate resistance genes.

The invention also concerns a process for the production of antibodies which is characterized in that a mammal is immunized with a carrier-bound recombinant protein which is obtainable by expression of a fusion protein in gram-negative bacteria and which contains at least one hydrophobic non-lytically active protein domain capable of membrane integration as well as the recombinant protein, if desired, with a delayed expression of a lytically active membrane protein from bacteriophages or of a lytically active toxin release gene or lytically active partial sequences thereof and the antibodies are obtained from the serum or the spleen according to well-known methods.

In a preferred embodiment B lymphocytes of the immunized animals are fused with a suitable cell line in the presence of transforming agents, the cell line which produces the desired antibodies is cloned and cultured and the monoclonal antibodies are isolated from the cells or the culture supernatant.

It has turned out that the process according to the present invention is particularly suitable for the production of viral immunogens such as e.g. HIV immunogens, HBV immunogens.

In addition, it has surprisingly turned out that the activity and thus the antigenic structures of recombinant antigens, which are usually obtained in an inactive form as refractile bodies (e.g. human proteins such as TPA or G-CSF) when expressed in prokaryotes, are preserved when expressed according to the process according to the present invention. The process according to the present invention therefore proves to be particularly advantageous for the production of immunogenic recombinant human proteins.

The present invention also concerns the use of the carrier-bound recombinant proteins according to the present invention as vaccines and for the stimulation of T lymphocytes.

The vaccines according to the present invention can be produced and used in the usual manner.

The present invention also concerns a process for the production of vaccines using the carrier-bound recombinant proteins according to the present invention. The production of these vaccines can be carried out according to the well-known methods. However, the carrier-bound recombinant protein is preferably first lyophilised and subsequently suspended, if desired, with addition of auxiliary substances.

Furthermore, it is preferred to formulate the vaccine as a multivalent vaccine. For this the carrier-bound recombinant protein according to the present invention can contain several recombinant antigens immobilized on the membrane of the bacterial ghost.

The vaccination with the vaccine according to the present invention can be carried out according to methods which are familiar to those skilled in the art, for example intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, orally and intranasally.

For the intramuscular or subcutaneous administration, the vaccine can for example be suspended in physiological saline. For the intranasal or intra-ocular application the vaccine can for example be applied in the form of a spray or an aqueous solution. For local, for example oral, administration it is often necessary to protect the immunogens temporarily against inactivation, for example against saccharolytic enzymes in the cavity of the mouth or against proteolytic enzymes in the stomach. Such a temporary protection can for example be effected by encapsulation of the immunogens. This encapsulation can for example be effected by coating with a protective agent (microencapsulation) or by embedding a multitude of immunogens according to the present invention in a protective carrier (macroencapsulation).

The encapsulation material can be semi-permeable or can become semi-permeable when introduced into the human or animal body. A biologically degradable substance is usually used as the carrier for the encapsulation.

The following examples figures and sequence protocols elucidate the invention further.

FIG. 1 shows diagrams of the plasmids pkSELS, pMLl and pMTVl

FIG. 2a-2d Diagram of a bacterial ghost as a carrier for recombinant proteins

a) longitudinal section through a gram-negative bacterium (om: outer membrane; pp: periplasmic space; im: inner (cytoplasmic) membrane, cp: cytoplasm).

b) Formation of a transmembrane lysis tunnel.

c) Cytoplasm flowing out through the lysis tunnel.

d) Bacterial ghost with fusion proteins which are anchored in the cell wall complex via target sequences.

EXAMPLE 1

N-terminal membrane targeting for HIV 1 gp41.

A HIV 1 specific DNA fragment is isolated from plasmid pHF14 as a 1445 bp DNA fragment by partial digestion with HincII/PvuII. The fragment contains the complete sequence of gp41, (345 codons of gp41) linker sequences, the last 45 codons of gp120. It corresponds to the nucleotides 4 to 1448 from SEQ ID NO: 1.

After linearizing plasmid pKSEL5 (SEQ ID NO:6) with AccI and filling up the protruding DNA ends with Klenow polymerase the HIV1 specific DNA fragment is ligated with this linearized plasmid. The plasmid which formed is denoted pHIE1 and contains in frame a fusion of a partial sequence of the E gene (E' target sequence) of PhiX174 with the above-mentioned HIV1 fragment, in which the natural stop codon of the HIV1 env-gene is preserved.

EXAMPLE 2

N- as well as C-terminal membrane targeting of HIV1 gp41.

A 1059 bp HIV1 specific DNA fragment is isolated from the plasmid pHFl4 by digestion with HincII. This fragment contains linker sequences at the 5' side followed by 45 codons from gp120 as well as 301 codons from gp41. It corresponds to the nucleotides 4 to 1062 from SEQ ID NO:1. After linearizing plasmid pKSEL5 with AccI and filling up the protruding DNA ends with Klenow polymerase the HIV1 specific DNA fragment is ligated with this vector. The plasmid pHIE3 which formed contains an in frame fusion of a partial sequence of the E gene (E' target sequence) with a partial sequence of HIV and a partial sequence of the L gene (L' target sequence ).

EXAMPLE 3

C-terminal membrane targeting of HIV1 gp41.

A 1061 bp DNA fragment is isolated from plasmid pHF14 with SalI and HincII. This fragment contains linker sequences on the 5' side followed by 45 codons of gp120 as well as 301 codons of gp41. It corresponds to the nucleotides 2 to 1062 from SEQ ID NO: 1. After removing the E' sequence from the plasmid pKSEL5 by digestion with XhoI/AccI the protruding DNA ends of the vector and of the isolated HIV1 fragment are filled up with the aid of Klenow polymerase and, ligated. The plasmid pHIE5 which formed contains an in frame fusion of the first 5 codons of the lacZ gene, polylinker codons, gp120/gp41 codons and polylinker codons followed by the L' target sequence.

EXAMPLE 4

C-terminal membrane targeting of streptavidin.

The 498 bp XbaI fragment (FXaStrpA, nucleotide 2 to 499 of SEQ ID NO:2) from pFN6 filled up with Klenow polymerase is ligated into the filled up cleavage sites of the plasmid pKSEL5 from which the E' gene fragment was deleted by cleavage with HincII/XhoI. The plasmid obtained is denoted pAV5. This gives rise in the plasmid pAV5 to an in frame fusion of the first5 codons of the LacZ gene, 26 amino acid codons from the remaining polylinker sequence as well as of the amino acid sequences of the FXaStrpA part followed by the L' target sequence.

EXAMPLE 5

N-terminal membrane targeting of streptavidin.

The streptavidin gene extended on the 5' side by a factor Xa protease cleavage site is isolated as a 511 bp fragment from plasmid pFN6 after digestion with BamHI. It contains nucleotides 14 to 524 from SEQ ID NO:2. After filling up the ends with Klenow polymerase this DNA fragment is integrated into the filled up XbaI cleavage site of vector pKSEL5 between the E' and L' target sequences. An in frame gene fusion thereby results in plasmid pAV1 consisting of the E' target sequence and the FXaStrpA sequence. The stop codons occurring on the 3' side of streptavidin remain intact by the cloning which was carried out.

EXAMPLE 6

N- and C-terminal membrane targeting of streptavidin.

The stop codons 5'-TAATAA-3' which are located behind the streptavidin gene in plasmid pAV1 are removed by deletion of a 33 bp long DNA fragment which is produced by partial digestion with HincII and subsequent digestion with XbaI. The streptavidin-specific DNA sequence contains nucleotides 14 to 499 from SEQ ID NO:2. After filling up the plasmid ends with Klenow polymerase and religating, the L' target sequence included on the vector is fused in frame to the E' target sequence and the FXaStrpA sequence (plasmid pAV3). The corresponding gene product is thus provided with an N- as well as a C-terminal target sequence.

EXAMPLE 7

N-terminal membrane targeting of β-galactosidase.

A 3124 bp DNA fragment (SEQ ID NO:3) is isolated from the plasmid pMC1403 (J. Bacteriol. 143 (1980) 971-980) with the aid of PstI and DraI and ligated in the correct orientation into the PstI and NruI restriction sites of the plasmid pKSEL5. The plasmid pLZl which formed contains the first 54 codons of the E' target sequence, 13 linker codons and 1015 codons of the LacZ gene. The PstI/DraI fragment used for plasmid pLZ1 extends in the sequence protocol SEQ ID NO:3 from nucleotide 26 to 3149 inclusive and comprises 3124 bp.

EXAMPLE 8

N- and C-terminal membrane targeting of β-galactosidase.

The 3010 bp LacZ DNA fragment (PstI-EcoRI, nucleotides 26 to 3035 from SEQ ID NO: 3) is isolated from plasmid pMC1403 and is integrated in the correct orientation into the PstI/HindIII restriction site of pKSEL5 after filling up the EcoRI and HindIII ends. In the plasmid pLZ3 thus obtained this results in an in frame fusion of the E' target sequence with the LacZ gene and the L' target sequence.

EXAMPLE 9

C-terminal membrane targeting of β-galactosidase.

Plasmid pLZ3 is digested with EcoRI and partially digested with AccI. The E' target sequence is thereby removed. The fragment contains the nucleotides 29-3035 from SEQ ID NO:3 and is 3007 bp long (after filling up the EcoRI cleavage site). After filling up the protruding DNA ends of the vector and religating, the vector pLZ5 results in which a lacZ-L' fusion gene is present and the gene product of which has a C-terminal membrane target sequence.

EXAMPLE 10

Production of the carrier-bound recombinant proteins via the plasmids pMTVl (SEQ ID NO:4), pkSEL and pMLl (SEQ ID NO:5).

A lysis cassette is present on the plasmids pMTVl and pMLl consisting of the lambda cI857 repressor gene, the lambda promotor/operator system pR on the right side as well as the PhiXl74 lysis gene E. The integration of the foreign gene can be carried out in the multiple cloning site mcs 2 for pMTVl or pkSEL5 (FIG. 1). This is carried out in an analogous manner to that described in Examples 1-9.

EXAMPLE 11

Fermentation and lysis

The plasmid is integrated into E. coli K12 (DSM 2093) and the culture is grown in a shaking flask up to OD 0.8-1.2 at 600 nm whereby the expression of the lysis gene E is repressed by cI857 repressor molecules (Eur. J. Biochem. 180 (1989) 393 to 398). The expression of gene E by thermal inactivation of cI857 repressor molecules is carried out by increasing the temperature to 42° C. during the exponential growth phase of the bacteria. The lysis of E. coli caused by protein E starts between 10 and 30 min after increasing the temperature depending on the culture medium of the bacteria (total medium or minimum medium, under aeration in a shaking water bath). After a further 10 to 30 min the lysis is completed.

EXAMPLE 12

Modified protein E-lysis

The culture is as in Example 11 in which, however, the culture medium is made up to 0.2 mol/1 magnesium sulphate by adding magnesium sulphate solution 30 min prior to increasing the temperature from 28° C. to 42° C. This prevents the lysis of the bacteria despite the expression of gene E.

The cells are harvested by centrifugation 30 min after increasing the temperature. An instantaneous lysis of the cells is effected by resuspension of the cell pellet in low molar buffer (PBS, 1 mmol/1 phosphate buffer, 1 to 10 mmol/1 Tris-HCl pH 6-8) or water. The cell coats which are obtained in this process are denoted bacterial ghosts. Under these conditions, which correspond to a combination of protein E lysis and osmotic shock, a larger lysis structure is obtained in the bacteria. The morphology of the bacterial ghosts is also preserved to a large degree under these conditions.

The bacterial ghosts are washed 2× with PBS or 0.9% NaCl for purification (resuspending and centrifuging) and lyophilized.

EXAMPLE 13

Immunization

For the immunization, 10⁹ germs (corresponding to 1 mg dry weight of bacterial ghosts) per mouse are administered 4× intraperitoneally in 0.9% NaCl at monthly intervals. 8 days after the last immunization serum is obtained and the antibodies are isolated.

EXAMPLE 14

Binding of biotinylated HBc antigen

Bacterial ghosts produced according to example 4, into which streptavidin is integrated via target sequences, are lyophilized. 10 ml of a solution of 20 Ig/ml of a conjugate of hepatitis B core antigen and biotin (produced by reaction of HBcAg with N-hydroxysuccinimide-activated biotin) in 40 mmol/1 phosphate buffer, pH 7.4 is added to 1 mg of this lyophilisate, incubated for 30 min and subsequently washed several times with 40 mmol/1 phosphate buffer, pH 7.4. In this way a carrier-bound HBcAg immunogen is obtained which can be used for immunization and isolation of antibodies.

    __________________________________________________________________________     SEQUENCE LISTING                                                               (1) GENERAL INFORMATION:                                                       (iii) NUMBER OF SEQUENCES: 6                                                   (2) INFORMATION FOR SEQ ID NO: 1:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1451 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                       GTCGACCTGC AGGCATGCAAGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAAT60                TGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCC120                ACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAG CTTTG180               TTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACG240                GTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCT300                ATTGAGGGCCAACAGCATCTGTTGCAACTCAC AGTCTGGGGCATCAAGCAGCTCCAGGCA360               AGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGC420                TCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCT480                CTGGAACAGA TTTGGAATAACATGACCTGGATGGAGTGGGACAGAGAAATTAACAATTAC540               ACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAA600                GAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAA ATTGG660               CTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTT720                TTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAG780                ACCCACCTCCCAAACCCGAGGGGACCCGACAG GCCCGAAGGAATAGAAGAAGAAGGTGGA840               GAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCCTTAGCACTTATCTGGGAC900                GATCTGCGGAGCCTGTGCCTCTTCAGCTACCACCGCTTGAGAGACTTACTCTTGATTGTA960                ACGAGGATTG TGGAACTTCTGGGACGCAGGGGGTGGGAAGCCCTCAAATATTGGTGGAAT1020              CTCCTACAGTATTGGAGTCAGGAACTAAAGAATAGTGCTGTTAACTTGCTCAATGCCACA1080               GCTATAGCAGTAGCTGAGGGGACAGATAGGGTTATAGAATTAGTACAAGCAGCTT ATAGA1140              GCCATTCGCCACATACCTAGAAGAATAAGACAGGGCTTGGAAAGGATTTTGCTATAAGAT1200               GGGTGGCAAGTGGTCAAAAAGTAGTGTGGTTGGATGGCCTGCTGTAAGGGAAAGAATGAG1260               ACGAGCTGAGCCAGCAGCAGATGGGGTGGGAG CAGTATCTCGAGACCTAGAAAAACATGG1320              AGCAATCACAAGTAGCAATACAGCAGCTACCAATGCCGATTGTGCTTGGCTAGAAGCACA1380               AGAGGAGGAGGAGGTGGGTTTTCCAGTCACACCTCAGGTACCTTTAAGACCAATGACTTA1440               CAAGGCAGCT G1451                                                               (2) INFORMATION FOR SEQ ID NO: 2:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 525 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                       TCTAGAACTAGTG GATCCATCGAGGGTAGGTCTATGGACCCGTCCAAGGACTCCAAAGCT60                CAGGTTTCTGCAGCCGAAGCTGGTATCACTGGCACCTGGTATAACCAACTGGGGTCGACT120                TTCATTGTGACCGCTGGTGCGGACGGAGCTCTGACTGGCACCTACGAATCTGCGGTTGGT18 0               AACGCAGAATCCCGCTACGTACTGACTGGCCGTTATGACTCTGCACCTGCCACCGATGGC240                TCTGGTACCGCTCTGGGCTGGACTGTGGCTTGGAAAAACAACTATCGTAATGCGCACAGC300                GCCACTACGTGGTCTGGCCAATACGTTGGCGGTGCTGAGGCTCGT ATCAACACTCAGTGG360               CTGTTAACATCCGGCACTACCGAAGCGAATGCATGGAAATCGACACTAGTAGGTCATGAC420                ACCTTTACCAAAGTTAAGCCTTCTGCTGCTAGCATTGATGCTGCCAAGAAAGCAGGCGTA480                AACAACGGTAACCCTCTAGACGCTGTTC AGCAATAATAAGGATCC525                              (2) INFORMATION FOR SEQ ID NO: 3:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 3152 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                       ATGACCATGATTACGAATTGCTGCAGGTCGACG GATCCCGTCGTTTTACAACGTCGTGAC60                TGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGC120                TGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAAT180                GGCGAATGGCGCT TTGCCTGGTTTCCGGCACCAGAAGCGGTGCCGGAAAGCTGGCTGGAG240               TGCGATCTTCCTGAGGCCGATACTGTCGTCGTCCCCTCAAACTGGCAGATGCACGGTTAC300                GATGCGCCCATCTACACCAACGTAACCTATCCCATTACGGTCAATCCGCCGTTTGTTCCC 360               ACGGAGAATCCGACGGGTTGTTACTCGCTCACATTTAATGTTGATGAAAGCTGGCTACAG420                GAAGGCCAGACGCGAATTATTTTTGATGGCGTTAACTCGGCGTTTCATCTGTGGTGCAAC480                GGGCGCTGGGTCGGTTACGGCCAGGACAGTCGTTTGCCGT CTGAATTTGACCTGAGCGCA540               TTTTTACGCGCCGGAGAAAACCGCCTCGCGGTGATGGTGCTGCGTTGGAGTGACGGCAGT600                TATCTGGAAGATCAGGATATGTGGCGGATGAGCGGCATTTTCCGTGACGTCTCGTTGCTG660                CATAAACCGACTACACAAAT CAGCGATTTCCATGTTGCCACTCGCTTTAATGATGATTTC720               AGCCGCGCTGTACTGGAGGCTGAAGTTCAGATGTGCGGCGAGTTGCGTGACTACCTACGG780                GTAACAGTTTCTTTATGGCAGGGTGAAACGCAGGTCGCCAGCGGCACCGCGCCTTTCGGC840                 GGTGAAATTATCGATGAGCGTGGTGGTTATGCCGATCGCGTCACACTACGTCTGAACGTC900               GAAAACCCGAAACTGTGGAGCGCCGAAATCCCGAATCTCTATCGTGCGGTGGTTGAACTG960                CACACCGCCGACGGCACGCTGATTGAAGCAGAAGCCTGCGATGTCGGT TTCCGCGAGGTG1020              CGGATTGAAAATGGTCTGCTGCTGCTGAACGGCAAGCCGTTGCTGATTCGAGGCGTTAAC1080               CGTCACGAGCATCATCCTCTGCATGGTCAGGTCATGGATGAGCAGACGATGGTGCAGGAT1140               ATCCTGCTGATGAAGCAGAACAACTTTA ACGCCGTGCGCTGTTCGCATTATCCGAACCAT1200              CCGCTGTGGTACACGCTGTGCGACCGCTACGGCCTGTATGTGGTGGATGAAGCCAATATT1260               GAAACCCACGGCATGGTGCCAATGAATCGTCTGACCGATGATCCGCGCTGGCTACCGGCG1320               ATGAGCGA ACGCGTAACGCGAATGGTGCAGCGCGATCGTAATCACCCGAGTGTGATCATC1380              TGGTCGCTGGGGAATGAATCAGGCCACGGCGCTAATCACGACGCGCTGTATCGCTGGATC1440               AAATCTGTCGATCCTTCCCGCCCGGTGCAGTATGAAGGCGGCGGAGCCGACACCA CGGCC1500              ACCGATATTATTTGCCCGATGTACGCGCGCGTGGATGAAGACCAGCCCTTCCCGGCTGTG1560               CCGAAATGGTCCATCAAAAAATGGCTTTCGCTACCTGGAGAGACGCGCCCGCTGATCCTT1620               TGCGAATACGCCCACGCGATGGGTAACAGTCTTGG CGGTTTCGCTAAATACTGGCAGGCG1680              TTTCGTCAGTATCCCCGTTTACAGGGCGGCTTCGTCTGGGACTGGGTGGATCAGTCGCTG1740               ATTAAATATGATGAAAACGGCAACCCGTGGTCGGCTTACGGCGGTGATTTTGGCGATACG1800               CCGAACGATCGCCAG TTCTGTATGAACGGTCTGGTCTTTGCCGACCGCACGCCGCATCCA1860              GCGCTGACGGAAGCAAAACACCAGCAGCAGTTTTTCCAGTTCCGTTTATCCGGGCAAACC1920               ATCGAAGTGACCAGCGAATACCTGTTCCGTCATAGCGATAACGAGCTCCTGCACTGGATG19 80              GTGGCGCTGGATGGTAAGCCGCTGGCAAGCGGTGAAGTGCCTCTGGATGTCGCTCCACAA2040               GGTAAACAGTTGATTGAACTGCCTGAACTACCGCAGCCGGAGAGCGCCGGGCAACTCTGG2100               CTCACAGTACGCGTAGTGCAACCGAACGCGACCGCATGGTCA GAAGCCGGGCACATCAGC2160              GCCTGGCAGCAGTGGCGTCTGGCGGAAAACCTCAGTGTGACGCTCCCCGCCGCGTCCCAC2220               GCCATCCCGCATCTGACCACCAGCGAAATGGATTTTTGCATCGAGCTGGGTAATAAGCGT2280               TGGCAATTTAACCGCCAGTCAG GCTTTCTTTCACAGATGTGGATTGGCGATAAAAAACAA2340              CTGCTGACGCCGCTGCGCGATCAGTTCACCCGTGCACCGCTGGATAACGACATTGGCGTA2400               AGTGAAGCGACCCGCATTGACCCTAACGCCTGGGTCGAACGCTGGAAGGCGGCGGGCCAT2460               TA CCAGGCCGAAGCAGCGTTGTTGCAGTGCACGGCAGATACACTTGCTGATGCGGTGCTG2520              ATTACGACCGCTCACGCGTGGCAGCATCAGGGGAAAACCTTATTTATCAGCCGGAAAACC2580               TACCGGATTGATGGTAGTGGTCAAATGGCGATTACCGTTGATGTTGAAGT GGCGAGCGAT2640              ACACCGCATCCGGCGCGGATTGGCCTGAACTGCCAGCTGGCGCAGGTAGCAGAGCGGGTA2700               AACTGGCTCGGATTAGGGCCGCAAGAAAACTATCCCGACCGCCTTACTGCCGCCTGTTTT2760               GACCGCTGGGATCTGCCATTGTCAGACATG TATACCCCGTACGTCTTCCCGAGCGAAAAC2820              GGTCTGCGCTGCGGGACGCGCGAATTGAATTATGGCCCACACCAGTGGCGCGGCGACTTC2880               CAGTTCAACATCAGCCGCTACAGTCAACAGCAACTGATGGAAACCAGCCATCGCCATCTG2940               CTGCACGCGG AAGAAGGCACATGGCTGAATATCGACGGTTTCCATATGGGGATTGGTGGC3000              GACGACTCCTGGAGCCCGTCAGTATCGGCGGAATTCCAGCTGAGCGCCGGTCGCTACCAT3060               TACCAGTTGGTCTGGTGTCAAAAATAATAATAACCGGGCAGGCCATGTCTGCCCGTA TTT3120              CGCGTAAGGAAATCCATTATGTACTATTTAAA3152                                           (2) INFORMATION FOR SEQ ID NO: 4:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 5314 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                       AAATTGTAAACGTTAATATTAGACATAATTTATCCTCAAGTAAGGGGCCGAAGCCCCTGC60                 AATTAAAATTGTTGACCACCTACATACCAAAGACGAGCGCCTTTACGCTTGCCTTTAGTA120                CCTCGCAACGGCTGCGGACGACCAGGGCGAGCGCCAGAACG TTTTTTACCTTTAGACATT180               ACATCACTCCTTCCGCACGTAATTTTTGACGCACGTTTTCTTCTGCGTCAGTAAGAACGT240                CAGTGTTTCCTGCGCGTACACGCAAGGTAAACGCGAACAATTCAGCGGCTTTAACCGGAC300                GCTCGACGCCATTAATAATGT TTTCCGTAAATTCAGCGCCTTCCATGATGAGACAGGCCG360               TTTGAATGTTGACGGGATGAACATAATAAGCAATGACGGCAGCAATAAACTCAACAGGAG420                CAGGAAAGCGAGGGTATCCCACAAAGTCCAGCGTACCATAAACGCAAGCCTCAACGCAGC480                G ACGAGCACGAGAGCGGTCAGTAGCAATCCAAACTTTGTTACTCGTCAGAAAATCGAAAT540               CATCTTCGGTTAAATCCAAAACGGCAGAAGCCTGAATTCTAGCTAGAGGATCTTTAGCTG600                TCTTGGTTTGCCCAAAGCGCATTGCATAATCTTTCAGGGTTATGCGTTG TTCCATACAAC660               CTCCTTAGTACATGCAACCATTATCACCGCCAGAGGTAAAATAGTCAACACGCACGGTGT720                TAGATATTTATCCCTTGCGGTGATAGATTTAACGTATGAGCACAAAAAAGAAACCATTAA780                CACAAGAGCAGCTTGAGGACGCACGTCGC CTTAAAGCAATTTATGAAAAAAAGAAAAATG840               AACTTGGCTTATCCCAGGAATCTGTCGCAGACAAGATGGGGATGGGGCAGTCAGGCGTTG900                GTGCTTTATTTAATGGCATCAATGCATTAAATGCTTATAACGCCGCATTGCTTACAAAAA960                TTCTCAAAG TTAGCGTTGAAGAATTTAGCCCTTCAATCGCCAGAGAAATCTACGAGATGT1020              ATGAAGCGGTTAGTATGCAGCCGTCACTTAGAAGTGAGTATGAGTACCCTGTTTTTTCTC1080               ATGTTCAGGCAGGGATGTTCTCACCTAAGCTTAGAACCTTTACCAAAGGTGATGCG GAGA1140              GATGGGTAAGCACAACCAAAAAAGCCAGTGATTCTGCATTCTGGCTTGAGGTTGAAGGTA1200               ATTCCATGACCGCACCAACAGGCTCCAAGCCAAGCTTTCCTGACGGAATGTTAATTCTCG1260               TTGACCCTGAGCAGGCTGTTGAGCCAGGTGATTTCT GCATAGCCAGACTTGGGGGTGATG1320              AGTTTACCTTCAAGAAACTGATCAGGGATAGCGGTCAGGTGTTTTTACAACCACTAAACC1380               CACAGTACCCAATGATCCCATGCAATGAGAGTTGTTCCGTTGTGGGGAAAGTTATCGCTA1440               GTCAGTGGCCTGAAGA GACGTTTGGCTGATCGGCAAGGTGTTCTGGTCGGCGCATAGCTG1500              ATAACAATTGAGCAAGAATCTTCATCGAATTAGGGGAATTTTCACTCCCCTCAGAACATA1560               ACATAGTAAATGGATTGAATTATGAAGAATGGTTTTTATGCGACTTACCGCAGCAAAAAT162 0              AAAGGGAAAGATACTTGAAGACGAAAGGGCATTTTGTTAAAATTCGCGTTAAATTTTTGT1680               TAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAA1740               GAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACA AGAGTCCACTATTAAAG1800              AACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGT1860               GAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAAC1920               CCTAAAGGGAGCCCCCGATTTAG AGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAG1980              GAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTG2040               CGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCA2100               TTC AGGCTACGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAG2160              CTGGCGAAGGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAG2220               TCACGACGTTGTAAAACGACGGCCAGTGAATTGTAATACGACTCACTATA GGGCGAATTG2280              GAGCTCCACCGCGGTGGCGGCCGCTCTAGTATGGTGCACTCTCAGTACAATCTGCTCTGA2340               TGCCGCATAGTTAAGCCAGTATATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTG2400               CGCCCCGACACCCGCCAACACCCGCTGACG CGCCCTGACGGGCTTGTCTGCTCCCGGCAT2460              CCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGT2520               CATCACCGAAACGCGCGAGGCAGTAAGGTCGGATGCTTTGTGAGCAATTCGTCCCTTAAG2580               TAAGCAATTG CTGTAAAGTCGTCACTGTGCGGATCACCGCTTCCAGTAGCGACAGAAGCA2640              ATTGATTGGTAAATTTCGAGAGAAAGATCGCGAGGAAGATCAATACATAAAGAGTTGAAC2700               TTCTTTGTTGTCTTCGACATGGGTAATCCTCATGTTTGAATGGCCCTAGAGGATCCGG CC2760              AAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGACGCTCGACGCCATTAATAAT2820               GTTTTCCGTAAATTCAGCGCCTTCCATGATGAGACAGGCCGTTTGAATGTTGACGGGATG2880               AACATAATAAGCAATGACGGCAGCAATAAACTCAACAG GAGCAGGAAAGCGAGGGTATCC2940              CACAAAGTCCAGCGTACCATAAACGCAAGCCTCAACGCAGCGACGAGCACGAGAGCGGTC3000               AGTAGCAATCCAAACTTTGTTACTCGTCAGAAAATCGAAATCATCTTCGGTTAAATCCAA3060               AACGGCAGAAGCCTGAAT GAGAATTCGACCTCGAGGGGGGGCCCGGTACCCAGCTTTTGT3120              TCCCTTTAGTGAGGGTTAATTCCGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTG3180               TGAAATTGTTATCCGCTCACAATTCCACACAACATAGGAGCCGGAAGCATAAAGTGTAAA3240               GCCTGGGGTGCCTAATGAGTGAGGTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCT3300               TTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGA3360               GGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGAC TCGCTGCGCTCGGTC3420              GTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAA3480               TCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGT3540               AAAAAGGCCGCGTTGCTGGCGTTTT TCCATAGGCTCGGCCCCCCTGACGAGCATCACAAA3600              AATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTC3660               CCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTG3720               TCCGC CTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTC3780              AGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCC3840               GACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGAC ACGACTTA3900              TCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCT3960               ACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATC4020               TGCGCTCTGCTGAAGCCAGTTACCTTCGGAAA AAGAGTTGGTAGCTCTTGATCCGGCAAA4080              CAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAA4140               AAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAA4200               AACTCACGTTAA GGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTT4260              TTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGAC4320               AGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCC 4380              ATAGTTGCCTGACTGCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGC4440               CCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATA4500               AACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTG CAACTTTATCCGCCTCCATC4560              CAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGC4620               AACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCA4680               TTCAGCTCCGGTTCCCAACG ATCAAGGCGAGTTACATGATCCCCCATGTTGTGAAAAAAA4740              GCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCA4800               CTCATGCTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTT4860                TCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGT4920              TGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTG4980               CTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTT ACCGCTGTTGAGA5040              TCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACC5100               AGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCG5160               ACACGGAAATGTTGAATACTCATACTC TTCCTTTTTCAATATTATTGAAGCATTTATCAG5220              GGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGG5280               GTTCCGCGCACATTTCCCCGAAAAGTGCCACCTG5314                                         (2) INFORMATION FOR SEQ ID NO: 5:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 7641 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:                                       GACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAG60                 TACTCACCAGT CACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGT120               GCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGA180                CCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCG T240               TGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCA300                GCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGG360                CAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCA GGACCACTTCTGCGCTCGGCC420               CTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGT480                ATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACG540                GGGAGTCAGGCAACTATGG ATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTG600               ATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAA660                CTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAA720                ATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCTTAATAAGATGATCTTCT780                TGAGATCGTTTTGGTCTGCGCGTAATCTCTTGCTCTGAAAACGAAAAAACCGCCTTGCAG840                GGCGGTTTTTCGAAGGTTCTCTGAGCTACCAACTCTTTGAACCGAG GTAACTGGCTTGGA900               GGAGCGCAGTCACCAAAACTTGTCCTTTCAGTTTAGCCTTAACCGGCGCATGACTTCAAG960                ACTAACTCCTCTAAATCAATTACCAGTGGCTGCTGCCAGTGGTGCTTTTGCATGTCTTTC1020               CGGGTTGGACTCAAGACGATAGTTAC CGGATAAGGCGCAGCGGTCGGACTGAACGGGGGG1080              TTCGTGCATACAGTCCAGCTTGGAGCGAACTGCCTACCCGGAACTGAGTGTCAGGCGTGG1140               AATGAGACAAACGCGGCCATAACAGCGGAATGACACCGGTAAACCGAAAGGCAGGAACAG1200               GAGAGC GCACGAGGGAGCCGCCAGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTT1260              TCGCCACCACTGATTTGAGCGTCAGATTTCGTGATGCTTGTCAGGGGGGCGGAGCCTATG1320               GAAAAACGGCTTTGCCGCGGCCCTCTCACTTCCCTGTTAAGTATCTTCCTGGC ATCTTCC1380              AGGAAATCTCCGCCCCGTTCGTAAGCCATTTCCGCTCGCCGCAGTCGAACGACCGAGCGT1440               AGCGAGTCAGTGAGCGAGGAAGCGGAATATATCCTGTATCACATATTCTGCTGACGCACC1500               GGTGCAGCCTTTTTTCTCCTGCCACATGAAGCA CTTCACTGACACCCTCATCAGTGCCAA1560              CATAGTAAGCCAGTATACACTCCGCTAGCGCTGAGGTCTGCCTCGTGAAGAAGGTGTTGC1620               TGACTCATACCAGGCCTGAATCGCCCCATCATCCAGCCAGAAAGTGAGGGAGCCACGGTT1680               GATGAGAGCTTTG TTGTAGGTGGACCAGTTGGTGATTTTGAACTTTTGCTTTGCCACGGA1740              ACGGTCTGCGTTGTCGGGAAGATGCGTGATCTGATCCTTCAACTCAGCAAAAGTTCGATT1800               TATTCAACAAAGCCACGTTGTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAA 1860              TATATCATCATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGTTAT1920               GAGCCATATTCAACGGGAAACGTCTTGCTCGAGGCCGCGATTAAATTCCAACATGGATGC1980               TGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGG CAATCAGGTGCGACAATCTA2040              TCGATTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGT2100               TGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCT2160               TCCGACCATCAAGCATTTTA TCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGAT2220              CCCCGGGAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGT2280               TGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTT2340                TAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGT2400              TGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGA2460               AATGCATAAGCTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGG TGATTTCTCACT2520              TGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGG2580               AATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCC2640               TTCATTACAGAAACGGCTTTTTCAAAAA TATGGTATTGATAATCCTGATATGAATAAATT2700              GCAGTTTCATTTGATGCTCGATGAGTTTTTCTAATCAGAATTGGTTAATTGGTTGTAACA2760               CTGGCAGAGCATTACGCTGACTTGACGGGACGGCGGCTTTGTTGAATAAATCGAACTTTT2820               GCTGAGTT GAAGGATCAGATCACGCATCTTCCCGACAACGCAGACCGTTCCGTGGCAAAG2880              CAAAAGTTCAAAATCACCAACTGGTCCACCTACAACAAAGCTCTCATCAACCGTGGCTCC2940               CTCACTTTCTGGCTGGATGATGGGGCGATTCAGGCCTGGTATGAGTCAGCAACAC CTTCT3000              TCACGAGGCAGACCTCAGCGCTCAAAGATGCAGGGGTAAAAGCTAACCGCATCTTTACCG3060               ACAAGGCATCCGGCAGTTCAACAGATCGGGAAGGGCTGGATTTGCTGAGGATGAAGGTGG3120               AGGAAGGTGATGTCATTCTGGTGAAGAAGCTCGAC CGTCTTGGCCGCGACACCGCCGACA3180              TGATCCAACTGATAAAAGAGTTTGATGCTCAGGGTGTAGCGGTTCGGTTTATTGACGACG3240               GGATCAGTACCGACGGTGATATGGGGCAAATGGTGGTCACCATCCTGTCGGCTGTGGCAC3300               AGGCTGAACGCCGGA GGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCA3360              GCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCA3420               AAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATAT34 80              TATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAG3540               AAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAA3600               GAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGT ATCACGAGGCCCTTTCGT3660              CTTCAAGTATCTTTCCCTTTATTTTTGCTGCGGTAAGTCGCATAAAAACCATTCTTCATA3720               ATTCAATCCATTTACTATGTTATGTTCTGAGGGGAGTGAAAATTCCCCTAATTCGATGAA3780               GATTCTTGCTCAATTGTTATCA GCTATGCGCCGACCAGAACACCTTGCCGATCAGCCAAA3840              CGTCTCTTCAGGCCACTGACTAGCGATAACTTTCCCCACAACGGAACAACTCTCATTGCA3900               TGGGATCATTGGGTACTGTGGGTTTAGTGGTTGTAAAAACACCTGACCGCTATCCCTGAT3960               CA GTTTCTTGAAGGTAAACTCATCACCCCCAAGTCTGGCTATGCAGAAATCACCTGGCTC4020              AACAGCCTGCTCAGGGTCAACGAGAATTAACATTCCGTCAGGAAAGCTTGGCTTGGAGCC4080               TGTTGGTGCGGTCATGGAATTACCTTCAACCTCAAGCCAGAATGCAGAAT CACTGGCTTT4140              TTTGGTTGTGCTTACCCATCTCTCCGCATCACCTTTGGTAAAGGTTCTAAGCTTAGGTGA4200               GAACATCCCTGCCTGAACATGAGAAAAAACAGGGTACTCATACTCACTTCTAAGTGACGG4260               CTGCATACTAACCGCTTCATACATCTCGTA GATTTCTCTGGCGATTGAAGGGCTAAATTC4320              TTCAACGCTAACTTTGAGAATTTTTGTAAGCAATGCGGCGTTATAAGCATTTAATGCATT4380               GATGCCATTAAATAAAGCACCAACGCCTGACTGCCCCATCCCCATCTTGTCTGCGACAGA4440               TTCCTGGGAT AAGCCAAGTTCATTTTTCTTTTTTTCATAAATTGCTTTAAGGCGACGTGC4500              GTCCTCAAGCTGCTCTTGTGTTAATGGTTTCTTTTTTGTGCTCATACGTTAAATCTATCA4560               CCGCAAGGGATAAATATCTAACACCGTGCGTGTTGACTATTTTACCTCTGGCGGTGA TAA4620              TGGTTGCATGTACTAAGGAGGTTGTATGGAACAACGCATAACCCTGAAAGATTATGCAAT4680               GCGCTTTGGGCAAACCAAGACAGCTAAAGATCCTCTAGCTAGAATTCAGGCTTCTGCCGT4740               TTTGGATTTAACCGAAGATGATTTCGATTTTCTGACG AGTAACAAAGTTTGGATTGCTAC4800              TGACCGCTCTCGTGCTCGTCGCTGCGTTGAGGCTTGCGTTTATGGTACGCTGGACTTTGT4860               GGGATACCCTCGCTTTCCTGCTCCTGTTGAGTTTATTGCTGCCGTCATTGCTTATTATGT4920               TCATCCCGTCAACATTC AAACGGCCTGTCTCATCATGGAAGGCGCTGAATTTACGGAAAA4980              CATTATTAATGGCGTCGAGCGTCCGGTTAAAGCCGCTGAATTGTTCGCGTTTACCTTGCG5040               TGTACGCGCAGGAAACACTGACGTTCTTACTGACGCAGAAGAAAACGTGCGTCAAAAATT5100               ACGTGCGGAAGGAGTGATGTAATGTCTAAAGGTAAAAAACGTTCTGGCGCTCGCCCTGGT5160               CGTCCGCAGCCGTTGCGAGGTACTAAAGGCAAGCGTAAAGGCGCTCGTCTTTGGTATGTA5220               GGTGGTCAACAATTTTAATTGCAGGGGCTTCGGCCCCTTACTTG AGGATAAATTATGTCT5280              AATATTCAAACTGGCGCCGAGCGTATGCCGCATGACCTTTCCCATCTTGGCTTCCTTGCT5340               GGTCAGATTGGTCGTCTTATTACCATTTCAACTACTCCGGTTATCGCTGGCGACTCCTTC5400               GAGATGGACGCCGTTGGCGCTCTC CGTCTTTCTCCATTGCGTCGTGGCCTTGCTATTGAC5460              TCTACTGTAGACATTTTTACTTTTTATGTCCCTCATCGTCACGTTTATGGTGAACAGTGG5520               ATTAAGTTCATGAAGGATGGTGTTAATGCCACTCCTCTCCCGACTGTTAACACTACTGGT5580               TATA TTGACCATGCCGCTTTTCTTGGCACGATTAACCCTGATACCAATAAAATCCCTAAG5640              CATTTGTTTCAGGGTTATTTGAATATCTATAACAACTATTTTAAAGCGCCGTGGATGCCT5700               GACCGTACCGAGGCTAACCCTAATGAGAATTCTCATGTTTGACAGCTTATC ATCGATAAG5760              CTTTAATGCGGTAGTTTATCACAGTTAAATTGCTAACGCAGTCAGGCACCGTGTATGAAA5820               TCTAACAATGCGCTCATCGTCATCCTCGGCACCGTCACCCTGGATGCTGTAGGCATAGGC5880               TTGGTTATGCCGGTACTGCCGGGCCTCTTGC GGGATATCGTCCATTCCGACAGCATCGCC5940              AGTCACTATGGCGTGCTGCTAGCGCTATATGCGTTGATGCAATTTCTATGCGCACCCGTT6000               CTCGGAGCACTGTCCGACCGCTTTGGCCGCCGCCCAGTCCTGCTCGCTTCGCTACTTGGA6060               GCCACTATCGA CTACGCGATCATGGCGACCACACCCGTCCTGTGGATCCGGATCAGCAGG6120              TGGAAGAGGGACTGGATTCCAAAGTTCTCAATGCTGCTTGCTGTTCTTGAATGGGGGGTC6180               GTTGACGACGACATGGCTCGATTGGCGCGACAAGTTGCTGCGATTCTCACCAATAAAAA A6240              CGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGG6300               ATCGCCGGATCTGAATTGCTATGTTTAGTGAGTTGTATCTATTTATTTTTCAATAAATAC6360               AATTGGTTATGTGTTTTGGGGGCGATCGTGAGGCAAAGA AAACCCGGCGCTGAGGCCGGA6420              AGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTG6480               CGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGC6540               CAACGCGCGGGGAGAGGCG GTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAG6600              TGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCG6660               GTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTGACGGCGGGAT6720               ATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCG6780               CAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAG6840               CATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGT TGAAAACCGGACAT6900              GGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTT6960               ATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGC7020               GATTTGCTGGTGACCCAATGCGACCA GATGCTCCACGCCCAGTCGCGTACCGTCTTCATG7080              GGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAAC7140               ATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGAT7200               CAGCCC ACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCC7260              GCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAAT7320               CGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGC CAATCAG7380              CAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGC7440               CATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCAC7500               GCGGGAAACGGTCTGATAAGAGACACCGGCATA CTCTGCGACATCGTATAACGTTACTGG7560              TTTCACATTCACCACCCTGAATTGACTCTCTTCCGGCGCTATCATGCCATACCGCGAAAG7620               GTTTTGCGCCATTCGATGGTG7641                                                      (2) INFORMATION FOR SEQ ID NO: 6:                                               (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 3681 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:                                       AAATTGTAAACGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCAT60                 TTTTTAACCAATAGGCCG AAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGA120               TAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCA180                ACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCT240                AATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCC300                CCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAG360                CGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGC TGCGCGTAACCACCA420               CACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAGGCTACGCA480                ACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAGGGGG540                GATGTGCTGCAAGGCGATTAAGTTG GGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTA600               AAACGACGGCCAGTGAATTGTAATACGACTCACTATAGGGCGAATTGGAGCTCCACCGCG660                GTGGCGGCCGCTCTAGTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTA720                AGCCA GTATATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCC780               GCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACA840                AGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCA CCGAAACG900               CGCGAGGCAGTAAGGTCGGATGCTTTGTGAGCAATTCGTCCCTTAAGTAAGCAATTGCTG960                TAAAGTCGTCACTGTGCGGATCACCGCTTCCAGTAGCGACAGAAGCAATTGATTGGTAAA1020               TTTCGAGAGAAAGATCGCGAGGAAGATCAATA CATAAAGAGTTGAACTTCTTTGTTGTCT1080              TCGACATGGGTAATCCTCATGTTTGAATGGCCCTAGAGGATCCGGCCAAGCTTGCATGCC1140               TGCAGGTCGACTCTAGAGGATCCCCGACGCTCGACGCCATTAATAATGTTTTCCGTAAAT1200               TCAGCGCCTTCC ATGATGAGACAGGCCGTTTGAATGTTGACGGGATGAACATAATAAGCA1260              ATGACGGCAGCAATAAACTCAACAGGAGCAGGAAAGCGAGGGTATCCCACAAAGTCCAGC1320               GTACCATAAACGCAAGCCTCAACGCAGCGACGAGCACGAGAGCGGTCAGTAGCAATCCAA 1380              ACTTTGTTACTCGTCAGAAAATCGAAATCATCTTCGGTTAAATCCAAAACGGCAGAAGCC1440               TGAATGAGAATTCGACCTCGAGGGGGGGCCCGGTACCCAGCTTTTGTTCCCTTTAGTGAG1500               GGTTAATTCCGAGCTTGGCGTAATCATGGTCATAGCTGTT TCCTGTGTGAAATTGTTATC1560              CGCTCACAATTCCACACAACATAGGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCT1620               AATGAGTGAGGTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAA1680               ACCTGTCGTGCCAGCTGCAT TAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTA1740              TTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGC1800               GAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACG1860                CAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGT1920              TGCTGGCGTTTTTCCATAGGCTCGGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAA1980               GTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTC CCCCCTGGAAGCT2040              CCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCC2100               CTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGG2160               TCGTTCGCTCCAAGCTGGGCTGTGTGC ACGAACCCCCCGTTCAGCCCGACCGCTGCGCCT2220              TATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAG2280               CAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGA2340               AGTGGTG GCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGA2400              AGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTG2460               GTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGAT CTCAAG2520              AAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAG2580               GGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAAT2640               GAAGTTTTAAATCAATCTAAAGTATATATGAGTA AACTTGGTCTGACAGTTACCAATGCT2700              TAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGAC2760               TGCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAA2820               TGATACCGCGAGAC CCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCG2880              GAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATT2940               GTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCA3 000              TTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTT3060               CCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGAAAAAAAGCGGTTAGCTCCT3120               TCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTG TTATCACTCATGCTTATGG3180              CAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTG3240               AGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGG3300               CGTCAATACGGGATAATACCG CGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAA3360              AACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGT3420               AACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGT3480               G AGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTT3540              GAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCA3600               TGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGT TCCGCGCACAT3660              TTCCCCGAAAAGTGCCACCTG3681                                                  

We claim:
 1. Immunogen comprising a non-lytic fusion protein bound to a portion of a gram negative bacterial cell membrane, wherein said fusion protein comprises:(i) one hydrophobic, non-lytically active protein domain capable of integration into a gram negative bacterial cell membrane wherein said hydrophobic, non-lytically active protein domain is selected from the group consisting of: (a) amino acids 1 to 54 of protein E of phage Phix174, and (b) amino acids 21 to 75 of protein L of phage MS2, and (ii) a protein foreign to a gram negative bacteria in which said fusion protein is expressed.
 2. The immunogen of claim 1, wherein said at least one hydrophobic, non-lytically active protein domain and said foreign protein are linked by a hydrophilic amino acid sequence of from 2 to 100 amino acids.
 3. The immunogen of claim 1, wherein said hydrophic, non-lytically active protein domain and said protein foreign to said gram negative bacteria are linked to each other via from 10 to 16 amino acids which have a β pleated secondary structure.
 4. The immunogen bacteria of claim 1, wherein said protein foreign to said gram negative bacteria is antigenic.
 5. The immunogen of claim 1, further comprising a non-covalently bound binding partner bound to said foreign protein.
 6. The immunogen of claim 1, further comprising an additional substance bound to said binding partner.
 7. The immunogen of claim 5, wherein said protein foreign to said gram negative bacteria comprises the protein portion of streptavidin or avidin.
 8. The immunogen of claim 5, wherein said covalently bound binding partner is a biotinylated antigen. 